Construction Of C-met Specific CAR-T Cells And Its Killing Effects On NSCLC Cells In Vivo And In Vitro

Background:Non-small cell lung cancer(NSCLC)is one of the malignant tumors with the highest morbidity and mortality worldwide.Most patients are diagnosed when they are in the advanced stage,so traditional therapies cannot significantly prolong the life cycle of NSCLC patients;Chimeric antigen receptor T cell(CAR-T)technology enables T cells to precisely target tumors through DNA recombination,which has been achieved a huge breakthrough in recent years;Because cellular-mesenchymal epithelial transition factor(c-Met)is more common on the surface of a variety of solid tumor cells such as liver,ovary,stomach,lung,and less c-Met molecule were found in other tissues.c-Met is the specific molecule of application value in tumor targeted theraand less in other tissue.All these sugges CAR-T possess a variety of research potential on solid tumors.Objective:Engineered the CAR-T targeting c-Met,verify their ability to target NSCLC in vitro,and to use nude mouse of A549 cell transplantation tumor models to observe their effectiveness and safety in inhibiting A549 cell transplantation tumor in vivo.Methods:1.The CAR sequence containing the c-Met antibody sc Fv sefragment was inserted into the lentiviral plasmid.Electrophoresis the digested plasmid to detect the correctness of the target gene;Producted lentiviral and calculated lentiviral titer useing the number of transfected HEK293 cells by lentiviral after doubling dilution;Engineered the CAR-T by lentivirus infection,and use Retro Nectin to improve the infection efficiency.The amount of virus was instillment in the infection system according to MOI=5.The infection system:1×10~6/m L T cells,virus,and the pro-infection reagent trans A;The expression of the CAR sequence was verified by q PCR and western blot.The positive rate and cell subtypes of c-Met CAR-T were detected by flow cytometry.2.Validate the positive expression of c-Met protein in NSCLC cells A549 and H1975by flow cytometry,and the negative expression of c-Met protein in ovarian cancer cells A2780.Use the c-Met antigen provided by A549 cells to stimulate c-Met CAR-T,the CCK-8method was used to evaluate their expansion rate,and A2780 cells were used to establish a Non target control;The LDH release method was used to detect the killing effect on A549and H1975 of c-Met CAR-T and activated T cells in effective target ratios of 1:1,1:5,1:10and 1:20.A2780 cells were used to set up a Non target control;Under the stimulation of the target antigen,ELISA detects the release of the cytokines IL-2,TNF-αand IFN-γof c-Met CAR-T and activated T cells in target antigen stimulation.A2780 cells were used to set up a Non target control.3.Nude mice were injected subcutaneously A549 cells labeled with luciferase,3×10~6/mouse.After tumor formation,c-Met CAR-T,activated T cells and PBS were used in three separated groups for treatment.The tumor volume and weight measurement and tumor imaging in vivo were used to evaluate the inhibitory effect of c-Met CAR-T cells on A549cell transplantation tumor in vivo.HE staining was performed on four organs of nude mice:liver,spleen,lung,and kidney to assess whether c-Met CAR-T cells exhibit off-target toxicity in vivo;The tumor was removed and the CAR-T cells were tested by immunohistochemistry.Remove the tumor tissue,the expression of target antigen c-Met and proliferation proteins Ki67 in cancer tissues were assessed by immunohistochemical method and the apoptosis was detected by the Tunel method.Results:1.The second generation c-Met CAR lentiviral vector plasmid was constructed successfully,and detection of electrophoretic band after restriction enzyme digestion.The results showed that the position of the band conformed to the theoretical value;The gene sequencing result conformed to the originally designed sequence,and the lentivirus was successfully packaged;Western blot and q PCR results indicate that the CAR molecule is present in T cells transfected with lentivirus,c-Met CAR-T were successfully constructed.;The positive rate of c-Met CAR-T detected by flow cytometry was 38.4%,and the proportion of CD8~+T cells is up-regulated after lentivirus infection.2.It was verified by flow cytometry that NSCLC cells A549 and H1975 have over-expression of c-Met and can be used as target cells.Ovarian cancer cell A2780 c-Met protein expression is negative and can be used as a Non target control;The CCK-8experiment results suggest that the number of c-Met CAR-T cells increased faster than the control group under the stimulation of the target antigen(p<0.01);The LDH release experiment results suggests that the anti-tumor effect of c-Met CAR-T cells increases with the increase of the effective target ratio.When the effective target ratio is 20:1,c-Met CAR-T have the highest killing ratio to A549 and H1974 cells,the killing rate reached 43.58%and51.12%.The ELISA results show that when the presence of target cells,,c-Met CAR-T cells can release more IL-2,TNF-αand IFN-γcompared to activated T cells.There was no significant difference in the amount of cytokines released by c-Met CAR-T and activated T cells in non target group.3.c-Met CAR-T can significantly inhibit the growth of subcutaneous tumor tissue in nude mice,and have no obvious off-target toxicity to the liver,spleen,lung and kidney of nude mice.The A549 cell apoptosis caused by c-Met CAR-T in vivo could be confirmed by Tunel method.The results of immunohistochemistry showed that after c-Met CAR-T treatment,the expression of proliferation-related protein Ki67 and the target antigen c-Met decreased significantly.Conclusions:1.Design the second-generation c-Met CAR molecule and successfully constructed c-Met CAR-T cells that can stably express the CAR sequence.2.The results of in vitro experiment showed that c-Met CAR-T can targeted recognize c-Met-positive lung cancer cells,and have tumor-specific cytotoxicity.3.c-Met CAR-T cells can inhibited A549 cell transplantation tumor growth,and have no obvious off-target toxicity to normal organs.